Anselm Fabian Lowell Schneider

Die Superauflösungsmikroskopie in lebenden Zellen ist durch die Verfügbarkeit kleiner Molekülsonden, die nur gegen wenige Targets und genetisch kodierte Tags existieren, eingeschränkt. Hier erweitern wir die Anwendbarkeit von STED mithilfe zellgängiger, stark fluoreszierender Nanobodys für das intrazelluläre Targeting. Um helle Fluoreszenzsignale bei niedriger Konzentration zu gewährleisten, nutzten wir intramolekulare Photostabilisierung, indem wir einen Fluorophor zusammen mit dem Photostabilisator Trolox mittels EPL an den Nanobodys ligierten. Zudem sind diese semi‐synthetischen Nanobodys mit einem spaltbaren, zellpenetrierenden Peptid für ein effizientes Eindringen in die Zelle ausgestattet. Dadurch können wir sowohl GFP und mCherry als auch zwei endogene Zielmoleküle, die nuklearen Lamine und das DNA‐Replikations‐ und Reparaturprotein PCNA, mittels superauflösender Bildgebung visualisieren. Wir haben zudem die Zellteilung und DNA‐Replikation mittels konfokaler oder STED‐Mikroskopie überwacht und damit den Nutzen dieser Werkzeuge für die Funktionsanalyse demonstriert.

Super‐resolution microscopy in living cells can be restricted by the availability of small molecule probes, which only exist against few targets and genetically encoded tags. Here, we expand the applicability of live‐cell STED by engineering cell‐permeable and highly fluorescent nanobodies as intracellular targeting agents. To ensure bright fluorescent signals at low concentrations we used the concept of intramolecular photostabilization by ligating a fluorophore along with the photostabilizer trolox to the nanobody using expressed protein ligation (EPL). Furthermore, these semi‐synthetic nanobodies are equipped with a cleavable cell‐penetrating peptide for efficient cellular entry, which enables super‐resolution imaging of GFP and mCherry, as well as two endogenous targets, nuclear lamins and the DNA replication and repair protein PCNA. We monitored cell division and DNA replication via confocal and STED microscopy thus demonstrating the utility of these new intracellular tools for functional analysis.

Herein, the application of N‐hydroxysuccinimide‐modified phosphonamidate building blocks for the incorporation of cysteine‐selective ethynylphosphonamidates into lysine residues of proteins, followed by thiol addition with small molecules and proteins, is reported. It is demonstrated that the building blocks significantly lower undesired homo‐crosslinking side products that can occur with commonly applied succinimidyl 4‐(N‐maleimidomethyl)cyclohexane‐1‐carboxylate (SMCC) under physiological pH. The previously demonstrated stability of the phosphonamidate moiety additionally solves the problem of premature maleimide hydrolysis, which can hamper the efficiency of subsequent thiol addition. Furthermore, a method to separate the phosphonamidate enantiomers to be able to synthesize protein conjugates in a defined configuration has been developed. Finally, the building blocks are applied to the construction of functional antibody–drug conjugates, analogously to FDA‐approved, SMCC‐linked Kadcyla, and to the synthesis of a functional antibody–protein conjugate.

The delivery of entire functional proteins into living cells is a long-sought goal in science. Cyclic cell-penetrating peptides (cCPPs) have proven themselves to be potent delivery vehicles to carry proteins upon conjugation into the cytosol of living cells with immediate bioavailability via a non-endosomal uptake pathway. With this strategy, we pursue the cytosolic delivery of mCherry, a medium-sized fluorescent protein. Afterward, we achieve subcellular delivery of mCherry to different intracellular loci by genetic fusion of targeting peptides to the protein sequence. We show efficient transport into a membrane-bound compartment, the nucleus, as well as targeting of the actin cytoskeleton, marking one of the first ways to label actin fluorescently in genetically unmodified living cells. Furthermore, we demonstrate that only by conjugation of cCPPs via a disulfide bond, is flawless localization to the target area achieved. This finding underlines the importance of using a cCPP-based delivery vehicle that is cleaved inside cells, for the precise intracellular localization of a protein of interest.

Nanobodys sind ein modernes Mittel zur Erkennung und Modulation von Antigenen, die für herkömmliche Antikörper unzugänglich sind. Aufgrund ihrer kompakten Form und ihrer hohen Stabilität werden sie häufig in der Grundlagenforschung verwendet. In diesem Aufsatz werden zentrale Aspekte der Funktionalisierung von Nanobodys nebst ausgewählten Anwendungen in der Molekularbiologie dargestellt. Während ältere Konjugationsstrategien auf der zufälligen Modifikation natürlicher Aminosäuren beruhen, basieren neuere Methoden auf ortsspezifischer Modifikation mit funktionellen Einheiten. Solche Techniken umfassen chemoenzymatische Ansätze, Ligation exprimierter Proteine und die Unterdrückung des Amber‐Codons in Kombination mit bioorthogonalen Modifikationsstrategien. Mit einer ständig wachsenden Auswahl an Methoden aus der Proteinsynthese und ‐konjugation sind auch die Anwendungen auf dem Vormarsch. Diese reichen von hochentwickelter Bildgebung und Massenspektrometrie bis hin zum Einschleusen von Nanobodys in lebende Zellen, um intrazelluläre Antigene zu visualisieren und zu manipulieren.

Nanobodies can be seen as next‐generation tools for the recognition and modulation of antigens that are inaccessible to conventional antibodies. Due to their compact structure and high stability, nanobodies see frequent usage in basic research, and their chemical functionalization opens the way towards promising diagnostic and therapeutic applications. In this Review, central aspects of nanobody functionalization are presented, together with selected applications. While early conjugation strategies relied on the random modification of natural amino acids, more recent studies have focused on the site‐specific attachment of functional moieties. Such techniques include chemoenzymatic approaches, expressed protein ligation, and amber suppression in combination with bioorthogonal modification strategies. Recent applications range from sophisticated imaging and mass spectrometry to the delivery of nanobodies into living cells for the visualization and manipulation of intracellular antigens.

Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments. We used this strategy for the non-endocytic delivery of two recombinant nanobodies into living cells, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumour suppressor p53 to the nucleolus, and thereby allowed the detection of protein-protein interactions that involve these two proteins in living cells. Furthermore, cell-permeable nanobodies permitted the co-transport of therapeutically relevant proteins, such as Mecp2, into the cells. This technology constitutes a major step in the labelling, delivery and targeted manipulation of intracellular antigens. Ultimately, this approach opens the door towards immunostaining in living cells and the expansion of immunotherapies to intracellular antigen targets.